A culture model of development reveals multiple properties of RPE tight junctions.
OBJECTIVE
A culture model was used to examine the development of tight junctions in the retinal pigment epithelium (RPE).


METHODS
Chick RPE was isolated on embryonic day 7 (E7), E10 or E14 and cultured on laminin-coated filters. Barrier properties were stimulated with E14 retinal conditioned medium. Morphology was characterized by confocal microscopy. Permeability was determined by measuring the flux of horseradish peroxidase (HRP), radiolabeled inulin and mannitol, and the transepithelial electrical resistance (TER). Changes in the expression of ZO-1 and a related protein, ZO-1LP, were determined by immunoblotting.


RESULTS
RPE from each age formed epithelial monolayers of similar height, but the density of the cultures varied in parallel with density changes in vivo. The cultures appeared to regulate the permeability to ions and nonionic solutes independently. With embryonic age, there was a progressive decrease in permeability that first affected larger and then smaller tracers. Despite a small decrease in the permeability to mannitol, there was a large decrease in the permeability to ions. This suggests that in E14 cultures tight junctions discriminated by charge, as well as size. Although E14 retinal conditioned medium reduced the permeability to all solutes, it appeared to regulate size discrimination more than charge discrimination. Despite large effects on permeability, conditioned medium had no effect on the expression of ZO-1 or ZO-1LP.


CONCLUSIONS
The ability of tight junctions to discriminate on the basis of charge and size is regulated independently during development. The permeability of tight junctions cannot be predicted by the level of ZO-1 expression.
Ban
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Department of Surgery, Yale University, New Haven, CT 06520-8062, USA.
Rizzolo
L J
LJ
D000818:Animals; D002452:Cell Count / Q000187:drug effects; D002463:Cell Membrane Permeability / Q000187:drug effects; D002642:Chick Embryo; D017077:Culture Media, Conditioned / Q000494:pharmacology; D046508:Culture Techniques; D019084:Fluorescent Antibody Technique, Indirect; D018507:Gene Expression Regulation, Developmental; D006735:Horseradish Peroxidase / Q000493:pharmacokinetics; D007444:Inulin / Q000493:pharmacokinetics; D008353:Mannitol / Q000493:pharmacokinetics; D008565:Membrane Proteins / Q000378:metabolism; D010590:Phalloidine / Q000032:analysis; D010750:Phosphoproteins / Q000378:metabolism; D010857:Pigment Epithelium of Eye / Q000737:chemistry / Q000166:cytology / Q000196:embryology*; D019108:Tight Junctions / Q000502:physiology*; D013997:Time Factors; D062826:Zonula Occludens-1 Protein
D017077:Culture Media, Conditioned; D008565:Membrane Proteins; D010750:Phosphoproteins; D062826:Zonula Occludens-1 Protein; D010590:Phalloidine; D008353:Mannitol; D007444:Inulin; D006735:Horseradish Peroxidase